1) Field of the Invention
This invention relates to the solid phase synthesis of serine-containing polypeptides, such as LH-RH agonists and antagonists, by a temporary minimal protection procedure.
2) Description of Related Art
The decapeptide LH-RH (luteinizing hormone-releasing hormone) is a neurohormone produced in the hypothalamus, which stimulates the secretion of the pituitary hormones, LH (luteinizing hormone) and FSH (follicle-stimulating hormone), which in turn act on the gonads to stimulate the synthesis of steroid hormones. The pulsatile release of LH-RH, and the consequent release of LH and FSH, controls the reproductive cycle in mammals.
LH-RH analogs are nona- or decapeptides which are structurally related to LH-RH and exhibit biological activity similar to that of LH-RH by acting on the pituitary--gonadal axis in competition with naturally occurring LH-RH. The analogs are the subject of intensive clinical investigation due to their demonstrated ability to alleviate the symptoms of endometriosis, prostate cancer, precocious puberty, and other hormonally mediated disorders. While certain LH-RH analogs are currently available for therapeutic use, their synthesis is a complicated and, consequently, expensive procedure which necessarily increases their ultimate cost. LH-RH analogs are conventionally described as either agonists or antagonists, depending upon their mode of action.
The paradoxical ability of the agonists to suppress gonadal function at high doses has been exploited in the therapeutic arena by the development and marketing of such drugs as Synarel.RTM. nafarelin acetate nasal solution for the treatment of endometriosis. The amino acid sequence of nafarelin is as follows: ##STR1##
Nafarelin and other LH-RH agonists such as leuprorelin, buserelin, goserelin, histrelin, triptorelin and deslorelin all differ from naturally occurring LH-RH by replacement of at least the glycine residue at the 6-position with a D-amino acid. The synthetic agonists then have, in common with the naturally occurring hormone, histidine at position 2, serine at position 4, tyrosine at position 5, and arginine at position 8, all of which have reactive side chains which may present synthetic difficulties.
The LH-RH antagonists directly suppress gonadotropin secretion and are, therefore, desired for their immediate and profound effects. Generally, antagonism requires deletion or replacement of the histidyl residue at position 2 in the naturally occurring hormone. From the synthetic perspective, the deletion of histidine reduces the opportunities for undesired side reactions; however, the presence of arginine, serine and tyrosine still requires that special steps be taken to avoid side reactions.
LH-RH analogs may be synthesized by various methods, such as are taught by J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco, 1969; J. Meinenhofer, Hormonal Proteins and Peptides, Vol. 2, page 46, Academic Press (New York), 1973; and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press (New York), 1965. The methods may be broadly characterized as either solution phase or solid phase techniques. Both methods involve the sequential addition of amino acids to a growing peptide chain; however, the more rigorous conditions of solid phase synthesis generally require that any reactive side chains on the amino acids be protected during formation of the amide linkage. The side chain protecting groups may be removed concurrently with the cleavage of the completed polypeptide from the inert support on which it is made or in a separate deprotection step.
One particularly useful solid phase synthetic method for preparing LH-RH analogs is disclosed in Nestor et al., U.S. Pat. No. 4,234,571. In this commonly used approach, the .alpha.-amino (N.sup..alpha.) function of each amino acid is protected by an acid sensitive group, such as t-butoxycarbonyl (Boc); any reactive side chains, as are present on arginine, serine, histidine and tyrosine, are also protected with strongly bound groups which require treatment with hydrogen fluoride or similarly drastic procedures for their removal. This use of N.sup..alpha. -Boc protection and HF labile side chain protection, while adequate for the preparation of research quantities of peptides, is not entirely satisfactory for large scale production. The fully protected amino acids are expensive and require a separate step for deprotection. Further, the use of hydrogen fluoride for the final deprotection, in addition to posing serious environmental hazards, contributes to commercially unacceptable yield losses.
Alternate protocols are no more appealing. Tien et al., Pept. Chem. 375-379, T. Shiba and S. Sakakibara (Ed.), Protein Research Foundation, Osaka (1988), have reported the synthesis of LH-RH using tosyl protection on histidine and benzyl protection on tyrosine and serine. This approach, while avoiding the use of hydrogen fluoride, still requires a separate dehydrogenation step to remove the benzyl protecting groups, with some reduction of tryptophan occurring.
D. H. Coy et al., Int. J. Peptide Protein Res., 14, 339-343 (1979) report the synthesis of the LH-RH antagonists, [D-Phe.sup.2,D-Trp.sup.3, D-Phe.sup.6 ]LH-RH using a variety of side chain protection protocols, all of which require HF deprotection: providing benzyl side chain protection of serine only (with strong acid salt protection of arginine), tosyl side chain protection of arginine only, both serine and arginine side chain protection, and serine, arginine and tyrosine (with 2-bromobenzyloxycarbonyl) side chain protection. N.sup..alpha. -Boc protection was employed in all cases and removed between steps with 33% trifluoroacetic acid in methylene chloride. Hydrogen fluoride was used to cleave the crude peptide from its support and to remove the side chain protecting groups. An "unprotected" synthesis employing only salt protection of arginine (as Arg.HCl) was also conducted. All of the protected syntheses gave poorer yields than that of the unprotected side chain synthesis which did not require HF treatment.
Coy et al. further reported the synthesis of the LH-RH agonist [D-Leu.sup.6, desGly-NH.sub.2.sup.10 ]LH-RH ethylamide with dinitrophenyl side chain protection of histidine only, salt protection of arginine, and no HF treatment. The dinitrophenyl side chain protecting group was removed during cleavage of the peptide from its support with a solution of ethylamine in dimethylformamide. The yield from the histidine protected synthesis was 34% versus 24% for a fully protected, HF cleavage synthesis. No comparison with an unprotected synthesis was made.
The art suggests that, of the various minimal protection strategies, histidine-only protection may provide some improvement in yield over fully protected syntheses for certain LH-RH agonists; however, no particular benefit is associated with any of the reported side chain protection tactics for LH-RH antagonists.
While the ideal approach for eliminating the HF deprotection step may be to conduct an unprotected synthesis, lack of protection for histidine leads to excessive racemization. Following Coy et al., however, we have found that the use of histidine-only protection results in high levels of a bis-serine impurity. Significant improvement over the teachings of the art is needed in order to obtain a practicable minimal protection synthesis for LH-RH analogs that does not require an HF deprotection step yet provides protection for those groups which, if unprotected, will adversely affect the purity and yield of peptide.
The disclosures of the aforementioned patents and publications are incorporated by reference herein.